This project involves the molecular genetic analysis of the mechanism of action, specificity, and control of bacteriophage P22 antirepressor. This novel regulatory protein represents a higher order of complexity in mechanisms of regulation of gene expression, since it works by interacting with another regulatory protein, phage repressor. Synthesis of antirepressor is in turn under negative control by unknown mechanisms. The mechanism of action of antirepressor will be studied in a defined in vitro biochemical system using purified bacteriophage lambda repressor and purified P22 antirepressor. The kinetics and stoichiometry of the binding reaction between repressor and antirepressor will be determined. A genetic approach will be used to study the mechanisms by which antirepressor synthesis is controlled. Mutants defective in control of antirepressor synthesis have been isolated and will be characterized in order to determine the number of regulatory gene products involved and their mechanism of action.